The invention provides benzotriazine compounds having formula (I). The benzotriazine compounds of the invention are capable of inhibiting kinases, such members of the Src kinase family, and various other specific receptor and non-receptor kinases....http://www.google.com.au/patents/US7456176?utm_source=gb-gplus-sharePatent US7456176 - Benzotriazine inhibitors of kinases

The invention provides benzotriazine compounds having formula (I). The benzotriazine compounds of the invention are capable of inhibiting kinases, such members of the Src kinase family, and various other specific receptor and non-receptor kinases.

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Claims(34)

1. A compound of structure (I):

wherein each of A is independently selected from a group consisting of CH, N, NH, O, S, and a part of a ring fusion to form a second ring, where the second ring is an aromatic, a heteroaromatic, a bicyclic aromatic, a bicyclic aromatic heterocyclic ring, or a bicyclic with only the first ring being aromatic or heteroaromatic;

each of B is independently selected from a group consisting of CH, N, NH, O, S, and a part of a ring fusion to form a second ring, where the second ring is an aromatic, a heteroaromatic, a bicyclic aromatic, a bicyclic aromatic heterocyclic ring, or a bicyclic with only the first ring being aromatic or heteroaromatic;

R0 is selected from a group consisting of H and lower alkyl;

L is selected from a group consisting of a bond, and a substituted or unsubstituted alkyl, alkenyl, or alkynyl linking moiety;

R1 is selected from a group consisting of C(R′)3, OR′, N(R′)2, NR′C(O)R′, NR′C(O)O(R′), NR′C(O)N(R′)2, SR′, C(O)(O)R′, C(O), C(O)N(R′)2, SO3R′, OSO2R′, SO2R′, SOR′, S(O)N(R′)2, OS(O)(O)N(R′)2, S(O)(O)N(R′)2, S(O)N(R′)2, PO4R′, OPO2R′, PO3R′, PO2R′, and a 3-6 membered heterocycle with one or more heterocyclic atoms with each heteroatom independently being capable of carrying any R′ group on it, wherein R′ is selected from a group consisting of hydrogen, lower alkyl, alkyl-hydroxyl, thiol-alkyl, alkyl-thiol, aminoalkyl, alkylamino, branched alkyl, branched alkyl hydroxyl, branched thio-alkyl, branched alkyl-thiol, branched aminoalkyl, branched alkylamino, and a closed 3-6 membered carbocycle or heterocycle, with each heteroatom in the 3-6 membered heterocycle being capable of carrying any R′ group on it, and wherein each R′ is independent in case there is more than one R′;

R2 is a substituent situated at position 5,6 or 8 of the ring, wherein R2 is selected from a group consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, iso-pentyl, phenyl, substituted phenyl, halogen, branched or unbranched alkylamino, branched or unbranched aminoalkyl, branched or unbranched alkyloxo, branched or unbranched oxyalkyl, branched or unbranched thioalkyl, branched or unbranched alkylthiol, CF3, sulfonamido, substituted sulfonamido, sulfonate, sulfonate ester, phosphate, phosphate ester, phosphonate, phosphonate ester, carboxo, amido, ureido, substituted carboxo, substituted amido, substituted ureido, or a 3-6 membered carbocycle or heterocycle attached to positions 5, 6 or 8 directly or through group L, each heteroatom independently being capable of carrying any group R2, with the further proviso that either one, two or three sub stituents R2 are present in the ring, each of the substituents R2 being the same or different;

The present invention relates generally to the use of compounds to treat a variety of disorders, diseases and pathologic conditions and more specifically to the use of benzotriazine compounds to treat disorders.

BACKGROUND

c-Src plays a major role in the growth, progression and metastasis of a large number of cancers. c-Src can be the transforming element of the oncogenic Rous sarcoma retrovirus. Subsequently, it has been demonstrated that c-Src kinase can have the oncogenic potential. Gene knockout experiments suggest that inhibition of some members of the Src family might have potential therapeutic benefit.

Tyrosine kinases (TKs) phosphorylate tyrosine residues in peptides and proteins. These enzymes are key elements in the regulation of cell signaling including cell proliferation and cell differentiation. Protein TKs comprise the receptor TKs, including the epidermal growth family members (HER1 and HER2 for example), platelet derived growth factor (PDGF) and kinases that play a role in angiogenesis (Tie-2 and KDR for example), and the cellular or non-receptor kinases, which include members of the Src family.

c-Src TK is one of three members of the Src family expressed ubiquitously. c-Src is expressed at low levels in most cell types and, in the absence of the appropriate extracellular stimuli, maintained in an inactive conformation through phosphorylation of a regulatory tyrosine domain at Tyr530. Activation of c-Src occurs through dephosphorylation of the Tyr530 site and phosphorylation of a second tyrosine, Tyr419, present in the kinase domain of the enzyme.

Src kinase modulates signal transduction through multiple oncogenic pathways, including EGFR, HER2, PDGFR, FGFR and VEGFR. Thus, blocking signaling through the inhibition of the kinase activity of Src can be an effective means of modulating aberrant pathways that drive the oncogenic transformation of cells.

There exists a body of evidence of misregulated increased kinase activity of c-Src in several human tumor types, most notably colon and breast tumors. Misregulated c-Src TK activity has also been associated with adhesion and cytoskeletal changes both in tumor cells and otherwise, ultimately resulting in an invasive phenotype that may be motile. c-Src TK activity has been shown to be an important component in the epithelial to mesenchymal transition that occurs in the early stages of invasion of carcinoma cells. c-Src activity is also known to be essential in the turnover of local adhesions, a critical cell-motility component. In in vivo models of metastases, c-Src inhibition markedly reduces the rate of lymph and liver metastases. Clinical data supports the link between misregulated Src activity and the increased invasive potential of tumor cells. In colon tumors, increased c-Src TK activity has been shown to correlate to tumor progression, with the highest activity found in metastatic tissue. Increased Src activity in colon tumors might be an indicator of poor prognosis. In breast and ovarian cancers, enhancement of Src kinase activity has been reported, and in transitional cell carcinoma of the bladder, c-Src activity peaked as superficial tumors became muscle invasive.

Biochemically, cellular stimuli that lead to Src activation result in increased association between Src and the cytoskeleton. As a result, Src mediates the phosphorylation of many intracellular substrates such as EGFR, FAK, PYK2, paxillin, Stat3, and cyclin D. The biological effects of these interactions affect cell motility, adhesion, cell cycle progression, and apoptosis and might have some connection to the disease related effects stated above. Thus, Src plays a role in responses to regional hypoxia, limited nutrients, and internal cellular effects to self-destruct.

Increased c-Src TK activity results in breakdown of the E-cadherin-mediated epithelial cell-cell adhesion, which can be restored by Src inhibition. Intimate connections between increased VEGF activity, Src activity, and cellular barrier function related to vascular leak have been also demonstrated. Inhibition of Src results in decrease in vascular leak when exogenous VEGF is administered in in vivo studies. Examples where excessive vascular permeability leads to particularly deleterious effects include pulmonary edema, cerebral edema, and cardiac edema.

The cascade of events leading to loss of endothelial barrier function is complex and incompletely understood. Data support some role for kinases in this process. For example, VEGF-mediated edema has been shown to involve intracellular signaling by Src family kinases, protein kinase C, and Akt kinase. Rho-associated kinases have been linked to thrombin-mediated vascular leakage, and protein kinase C to TNF-induced leakage. Kinases are believed to mediate the phosphorylation of junctional proteins such as beta-catenin and vascular endothelial VE-cadherin, leading to the dissolution of adherens junctions and the dissociation of cadherin-catenin complexes from their cytoskeletal anchors. Proteins which regulate the intercellular contractile machinery such as myosin light chain kinase (MLCK) and myosin light chain (MLC) are also activated, resulting in cellular contraction, and therefore an opening of intercellular junctions.

A general approach to the inhibition of vascular leakage can be to interfere with any of the underlying mechanistic pathways, whether by inhibition of kinase signaling or the intercellular contractile apparatus or other cellular processes. This can then lead to potential treatments for edema and its associated pathologies. For example, inhibiting edema formation should be beneficial to overall patient outcome in situations such as inflammation, allergic diseases, cancer, cerebral stroke, myocardial infarction, pulmonary and cardiac insufficiency, renal failure, and retinopathies, to name a few. Furthermore, as edema is a general consequence of tissue hypoxia, it can also be concluded that inhibition of vascular leakage represents a potential approach to the treatment of tissue hypoxia. For example, interruption of blood flow by pathologic conditions (such as thrombus formation) or medical intervention (such as cardioplegia, organ transplantation, and angioplasty) could be treated both acutely and prophylactically using inhibitors of vascular leakage, especially as in the case of Src inhibitors.

Accordingly, a small molecule inhibitor of c-Src can be beneficial for the treatment of several disease states.

SUMMARY

The present invention provides methods of use for certain chemical compounds such as kinase inhibitors for treatment of various diseases, disorders, and pathologies, for example, cancer, and vascular disorders, such as myocardial infarction (MI), stroke, or ischemia.

The benzotriazine compounds described in this invention may block the enzymatic activity of some or many of the members of the Src family, in addition to blocking the activity of other receptor and non-receptor kinases. Such compounds may be beneficial for treatment of the diseases where disorders affect cell motility, adhesion, and cell cycle progression, and in addition, diseases with related hypoxic conditions, osteoporosis and conditions, which result from or are related to increases in vascular permeability, inflammation or respiratory distress, tumor growth, invasion, angiogenesis, metastases and apoptosis.

According to the embodiments of the invention, some examples of kinase inhibitors that can be used to bring about beneficial therapeutic results include inhibitors of Src kinase.

According to one embodiment of the invention, there are provided compounds having the structure (I), and pharmaceutically acceptable salts, hydrates, solvates, crystal forms, N-oxides, and individuals diastereoners thereof:

wherein each of A can be (CH)0-1, N, NH, O, S, or a part of a ring fusion to form a second ring, where the second ring can be an aromatic, a heteroaromatic, a bicyclic aromatic, a bicyclic aromatic heterocyclic ring, or a bicyclic with only the first ring being aromatic or heteroaromatic;

each of B can be (CH)0-1, N, NH, O, S, or a part of a ring fusion to form a second ring, where the second ring can be an aromatic, a heteroaromatic, a bicyclic aromatic, a bicyclic aromatic heterocyclic ring, or a bicyclic with only the first ring being aromatic or heteroaromatic, with the further proviso that if each B is (CH)0, R3 can be any substitutent described below, other than hydrogen, bonded directly to the position 7 of the adjacent ring;

R0 can be H or lower alkyl;

L can be a bond, or a substituted or unsubstituted alkyl, alkenyl, or alkynyl linking moiety;

R1 can be C(R′)3, OR′, N(R′)2, NR′C(O)R′, NR′C(O)O(R′), NR′C(O)N(R′)2, SR′, C(O)(O)R′, C(O), C(O)N(R′)2, SO3R′, OSO2R′, SO2R′, SOR′, S(O)N(R′)2, OS(O)(O)N(R′)2, S(O)(O)N(R′)2, S(O)N(R′)2, PO4R′, OPO2R′, PO3R′, PO2R′, or a 3-6 membered heterocycle with one or more heterocyclic atoms, with each heteroatom being capable of carrying any R′ group on it, wherein R′ can be hydrogen, lower alkyl, alkyl-hydroxyl, thiol-alkyl, alkyl-thiol, aminoalkyl, alkylamino, branched alkyl, branched alkyl hydroxyl, branched thio-alkyl, branched alkyl-thiol, branched aminoalkyl, branched alkylamino, or a closed 3-6 membered carbocycle or heterocycle, with each heteroatom in the 3-6 membered heterocycle being capable of carrying any R′ group on it, and wherein each R′ can be independent in case there is more than one R′;

R2 is a substitutent situated at position 5, 6 or 8 of the ring, wherein R2 can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, iso-pentyl, phenyl, substituted phenyl, halogen, branched or unbranched alkylamino, branched or unbranched aminoalkyl, branched or unbranched alkyloxo, branched or unbranched oxyalkyl, branched or unbranched thioalkyl, branched or unbranched alkylthiol, CF3, sulfonamido, substituted sulfonamido, sulfonate, sulfonate ester, phosphate, phosphate ester, phosphonate, phosphonate ester, carboxo, amido, ureido, substituted carboxo, substituted amido, substituted ureido, or a 3-6 membered carbocycle or heterocycle attached to positions 5, 6 or 8 directly or through group L, each heteroatom being capable of carrying any group R2, with the further proviso that either one, two or three substituents R2 can be present in the ring, and each of the substituents R2 can be the same or different;

n is an integer that can have value between 1 and 5, with the further proviso that if n≧2, then each group R3 is independent of the other groups R3,

with the further proviso that if each A is (CH)0, L is a bond.

In yet another embodiment, there are provided articles of manufacture including packaging material and a pharmaceutical composition contained within the packaging material, wherein the packaging material includes a label which indicates that the pharmaceutical composition can be used for treatment of disorders associated with compromised vasculostasis, and wherein the pharmaceutical composition includes at least one compound of structure (I).

In another embodiment, there are provided articles of manufacture including packaging material and a pharmaceutical composition contained within the packaging material, wherein the packaging material includes a label which indicates that the pharmaceutical composition can be used for treatment of disorders associated with vascular permeability leakage or compromised vasculostasis, such as myocardial infarction, stroke, congestive heart failure, an ischemia or reperfusion injury, cancer, arthritis or other arthropathy, retinopathy or vitreoretinal disease, macular degeneration, autoimmune disease, vascular leakage syndrome, inflammatory disease, edema, transplant rejection, burn, or acute or adult respiratory distress syndrome (ARDS) and wherein the pharmaceutical composition includes at least one compound of structure (I).

In another embodiment, there are provided methods of treating a disorder associated with compromised vasculostasis, including the administration of a therapeutically effective amount of at least one compound of structure (I) or pharmaceutically acceptable salts, hydrates, solvates, crystal forms and individual diastereomers thereof, to a subject in need of such treatment.

In yet another embodiment, there are provided methods of treating a disorder associated with compromised vasculostasis including the administration of a therapeutically effective amount of at least one compound of structure (I), or pharmaceutically acceptable salts, hydrates, solvates, crystal forms and individual diastereomers thereof, in combination with an anti-inflammatory, chemotherapeutic agent, immunomodulatory agent, therapeutic antibody or a protein kinase inhibitor, to a subject in need of such treatment.

In others embodiment, there are provided methods of treating a subject having or at risk of having a disorder selected from myocardial infarction, vascular leakage syndrome (VLS), cancer, stroke, ARDS, burns, arthritis, edema, retinopathy or vitreoretinal disease, ischemic or reperfusion related tissue injury or damage, autoimmune disease, transplant rejection, inflammatory disease, including administering to the subject a therapeutically effective amount of at least one compound of structure (I), thereby treating the subject.

In another embodiment, there are provided processes for making a pharmaceutical composition including combining a combination of at least one compound of structure (I) or its pharmaceutically acceptable salts, hydrates, solvates, crystal forms salts and individual diastereomers thereof and a pharmaceutically acceptable carrier.

DETAILED DESCRIPTIONA. Terms and Definitions

The following terminology and definitions apply as used in the present application, generally in conformity with the terminology recommended by the International Union of Pure and Applied Chemistry (IUPAC):

The term “heterocyclic,” when used to describe an aromatic ring, refer to the aromatic ring containing at least one heteroatom.

The term “heteroatom” refers to any atom other than carbon, for example, N, O, or S.

The term “aromatic” refers to a cyclically conjugated molecular entity with a stability, due to delocalization, significantly greater than that of a hypothetical localized structure, such as the Kekulé structure.

The term “heterocyclic,” when not used to describe an aromatic ring, refers to cyclic (i.e., ring-containing) groups other than aromatic groups, the cyclic group being formed by between 3 and about 14 carbon atoms and at least one heteroatom described above.

The term “substituted heterocyclic” refers, for both aromatic and non-aromatic structures, to heterocyclic groups further bearing one or more substituents described above.

The term “alkyl” refers to a monovalent straight or branched chain hydrocarbon group having from one to about 12 carbon atoms, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-pentyl (also known as n-amyl), n-hexyl, and the like.

The term “lower alkyl” refers to alkyl groups having from 1 to about 6 carbon atoms.

The term “alkenyl” refers to straight-chained or branched hydrocarbyl groups having at least one carbon-carbon double bond, and having between about 2 and about 12 carbon atoms, and the term “substituted alkenyl” refers to alkenyl groups further bearing one or more substituents described above.

The term “alkynyl” refers to straight-chained or branched hydrocarbyl groups having at least one carbon-carbon triple bond, and having between about 2 and about 12 carbon atoms, and the term “substituted alkynyl” refers to alkynyl groups further bearing one or more substituents described above.

The term “aryl” refers to aromatic groups having between about 5 and about 14 carbon atoms and the term “substituted aryl” refers to aryl groups further bearing one or more substituents described above.

The term “heteroaryl” refers to aromatic rings, where the ring structure is formed by between 3 and about 14 carbon atoms and by at least one heteroatom described above, and the term “substituted heteroaryl” refers to heteroaryl groups further bearing one or more substituents described above.

The term “alkoxy” refers to the moiety —O-alkyl, wherein alkyl is as defined above, and the term “substituted alkoxy” refers to alkoxy groups further bearing one or more substituents described above.

The term “cycloalkyl” refers to alkyl groups having between 3 and about 8 carbon atoms arranged as a ring, and the term “substituted cycloalkyl” refers to cycloalkyl groups further bearing one or more substituents described above.

The term “alkylaryl” refers to alkyl-substituted aryl groups and the term “substituted alkylaryl” refers to alkylaryl groups further bearing one or more substituents described above.

The term “arylalkyl” refers to aryl-substituted alkyl groups and the term “substituted arylalkyl” refers to arylalkyl groups further bearing one or more substituents described above.

The term “arylalkenyl” refers to aryl-substituted alkenyl groups and the term “substituted arylalkenyl” refers to arylalkenyl groups further bearing one or more substituents described above.

The term “arylalkynyl” refers to aryl-substituted alkynyl groups and the term “substituted arylalkynyl” refers to arylalkynyl groups further bearing one or more substituents described above.

The term “arylene” refers to divalent aromatic groups having between 5 and about 14 carbon atoms and the term “substituted arylene” refers to arylene groups further bearing one or more substituents described above.

The term “kinase” refers to any enzyme that catalyzes the addition of phosphate groups to a protein residue; for example, serine and threonine kinases catalyze the addition of phosphate groups to serine and threonine residues.

The terms “Src kinase,” “Src kinase family,” and “Src family” refer to the related homologs or analogs belonging to the mammalian family of Src kinases, including, for example, c-Src, Fyn, Yes and Lyn kinases and the hematopoietic-restricted kinases Hck, Fgr, Lck and Blk.

The terms “Src kinase signaling pathway,” and “Src cascade” refer to both the upstream and downstream components of the Src signaling cascade.

The term “therapeutically effective amount” refers to the amount of the compound or pharmaceutical composition that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician, e.g., restoration or maintenance of vasculostasis or prevention of the compromise or loss or vasculostasis; reduction of tumor burden; reduction of morbidity and/or mortality.

The term “pharmaceutically acceptable” refers to the fact that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The terms “administration of a compound” or “administering a compound” refer to the act of providing a compound of the invention or pharmaceutical composition to the subject in need of treatment.

The term “antibody” refers to intact molecules of polyclonal or monoclonal antibodies, as well as fragments thereof, such as Fab and F(ab′)2, Fv and SCA fragments which are capable of binding an epitopic determinant.

The term “vasculostasis” refers to the maintenance of the homeostatic vascular functioning leading to the normal physiologic functioning. The term “vasculostatic agents” refers to agents that seek to address conditions in which vasculostasis is compromised by preventing the loss of or restoring or maintaining vasculostasis.

B. Embodiments of the Invention

According to an embodiment of the invention, compounds having the structure (I) are provided for treatment of various diseases, disorders, and pathologies, as well as pharmaceutically acceptable salts, hydrates, solvates, crystal forms, N-oxides, and individuals diastereoners of compounds having the structure (I):

wherein each of A can be (CH)0-1, N, NH, O, S, or a part of a ring fusion to form a second ring, where the second ring can be an aromatic, a heteroaromatic, a bicyclic aromatic, a bicyclic aromatic heterocyclic ring, or a bicyclic with only the first ring being aromatic or heteroaromatic;

each of B can be (CH)0-1, N, NH, O, S, or a part of a ring fusion to form a second ring, where the second ring can be an aromatic, a heteroaromatic, a bicyclic aromatic, a bicyclic aromatic heterocyclic ring, or a bicyclic with only the first ring being aromatic or heteroaromatic, with the further proviso that if each B is (CH)0, R3 can be any substitutent described below, other than hydrogen, bonded directly to the position 7 of the adjacent ring;

R0 can be H or lower alkyl;

L can be a bond, or a substituted or unsubstituted alkyl, alkenyl, or alkynyl linking moiety;

R1 can be C(R′)3, OR′, N(R′)2, NR′C(O)R′, NR′C(O)O(R′), NR′C(O)N(R′)2, SR′, C(O)(O)R′, C(O), C(O)N(R′)2, SO3R′, OSO2R′, SO2R′, SOR′, S(O)N(R′)2, OS(O)(O)N(R′)2, S(O)(O)N(R′)2, S(O)N(R′)2, PO4R′, OPO2R′, PO3R′, PO2R′, or a 3-6 membered heterocycle with one or more heterocyclic atoms, with each heteroatom being capable of carrying any R′ group on it, wherein R′ can be hydrogen, lower alkyl, alkyl-hydroxyl, thiol-alkyl, alkyl-thiol, aminoalkyl, alkylamino, branched alkyl, branched alkyl hydroxyl, branched thio-alkyl, branched alkyl-thiol, branched aminoalkyl, branched alkylamino, or a closed 3-6 membered carbocycle or heterocycle, with each heteroatom in the 3-6 membered heterocycle being capable of carrying any R′ group on it, and wherein each R′ can be independent in case there is more than one R′;

R2 is a substitutent situated at position 5, 6 or 8 of the ring, wherein R2 can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, iso-pentyl, phenyl, substituted phenyl, halogen, branched or unbranched alkylamino, branched or unbranched aminoalkyl, branched or unbranched alkyloxo, branched or unbranched oxyalkyl, branched or unbranched thioalkyl, branched or unbranched alkylthiol, CF3, sulfonamido, substituted sulfonamido, sulfonate, sulfonate ester, phosphate, phosphate ester, phosphonate, phosphonate ester, carboxo, amido, ureido, substituted carboxo, substituted amido, substituted ureido, or a 3-6 membered carbocycle or heterocycle attached to positions 5, 6 or 8 directly or through group L, each heteroatom being capable of carrying any group R2, with the further proviso that either one, two or three substituents R2 can be present in the ring, and each of the substituents R2 can be the same or different;

Src-family tyrosine kinases other than Lck, such as Hck and Fgr, are important in the Fc gamma receptor induced respiratory burst of neutrophils as well as the Fc gamma receptor responses of monocytes and macrophages. The compositions and methods of the present invention may be useful in inhibiting the Fc gamma induced respiratory burst response in neutrophils, and may also be useful in inhibiting the Fc gamma dependent production of TNF alpha. The ability to inhibit Fc gamma receptor dependent neutrophil, monocyte and macrophage responses would result in additional anti-inflammatory activity for the compounds employed in invention methods. This activity would be especially of value, for example, in the treatment of inflammatory diseases, such as arthritis or inflammatory bowel disease. The compositions and methods of the present invention may also be useful in the treatment of autoimmune glomerulonephritis and other instances of glomerulonephritis induced by deposition of immune complexes in the kidney that trigger Fc gamma receptor responses and which can lead to kidney damage.

In addition, certain Src-family tyrosine kinases, such as Lyn and Src, may be important in the Fc epsilon receptor induced degranulation of mast cells and basophils that plays an important role in asthma, allergic rhinitis, and other allergic disease. Fc epsilon receptors are stimulated by IgE-antigen complexes. Compounds employed in the methods of the present invention may inhibit the Fc epsilon induced degranulation responses. The ability to inhibit Fc epsilon receptor dependent mast cell and basophil responses may result in additional anti-inflammatory activity for the present compounds beyond their effect on T cells.

The present invention also provides articles of manufacture comprising packaging material and a pharmaceutical composition contained within the packaging material, wherein the packaging material comprises a label which indicates that the pharmaceutical composition can be used for treatment of disorders and wherein the pharmaceutical composition comprises a compound according to the present invention. Thus, in one aspect, the invention provides a pharmaceutical composition including a therapeutic agent and a compound of the invention, wherein the compound is present in a concentration effective to reduce vascular leakage associated with indications or therapeutic agents which have vascular leak as a side effect. For example, administration of a compound of the invention can be in conjunction with IL-2, immunotoxins, antibodies or chemotherapeutics. In these cases, IL-2, immunotoxin, antibody or chemotherapeutic concentration can be determined by one having ordinary skill in the art according to standard treatment regimen or, for example, as determined by an in vivo animal assay.

The present invention also provides pharmaceutical compositions comprising IL-2, immunotoxin, antibody or chemotherapeutic and at least one invention compound in an amount effective for inhibiting vascular permeability, and a pharmaceutically acceptable vehicle or diluent. The compositions of the present invention may contain other therapeutic agents, and may be formulated, for example, by employing conventional solid or liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration (for example, excipients, binders, preservatives, stabilizers, flavors, etc.) according to techniques known in the art of pharmaceutical formulation.

The compounds of the invention may be formulated into therapeutic compositions as natural or salt forms. Pharmaceutically acceptable non-toxic salts include the base addition salts (formed with free carboxyl or other anionic groups) which may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine, procaine, and the like. Such salts may also be formed as acid addition salts with any free cationic groups and will generally be formed with inorganic acids such as, for example, hydrochloric, sulfuric, or phosphoric acids, or organic acids such as acetic, citric, p-toluenesulfonic, methanesulfonic acid, oxalic, tartaric, mandelic, and the like. Salts of the invention include amine salts formed by the protonation of an amino group with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like. Salts of the invention also include amine salts formed by the protonation of an amino group with suitable organic acids, such as p-toluenesulfonic acid, acetic acid, and the like. Additional excipients which are contemplated for use in the practice of the present invention are those available to those of ordinary skill in the art, for example, those found in the United States Pharmacopeia Vol. XXII and National Formulary Vol. XVII, U.S. Pharmacopeia Convention, Inc., Rockville, Md. (1989), the relevant contents of which is incorporated herein by reference. In addition, polymorphs of the invention compounds are included in the present invention.

Pharmaceutical compositions of the invention may be administered by any suitable means, for example, orally, such as in the form of tablets, capsules, granules or powders; sublingually; buccally; parenterally, such as by subcutaneous, intravenous, intramuscular, intrathecal, or intracisternal injection or infusion techniques (e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions); nasally such as by inhalation spray; topically, such as in the form of a cream or ointment; or rectally such as in the form of suppositories; in dosage unit formulations containing non-toxic, pharmaceutically acceptable vehicles or diluents. The present compounds may, for example, be administered in a form suitable for immediate release or extended release. Immediate release or extended release may be achieved by the use of suitable pharmaceutical compositions comprising the present compounds, or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps. The present compounds may also be administered liposomally.

In addition to primates, such as humans, a variety of other mammals can be treated according to the method of the present invention. For instance, mammals including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine, canine, feline, rodent or murine species can be treated. However, the method can also be practiced in other species, such as avian species (e.g., chickens).

The pharmaceutical compositions for the administration of the compounds of this embodiment either alone or in combination with IL-2, immunotoxin, antibody or chemotherapeutic may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical composition the active object compound is included in an amount sufficient to produce the desired effect upon the process or condition of diseases. The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.

Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated to form osmotic therapeutic tablets for control release.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. Also useful as a solubilizer is polyethylene glycol, for example. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a parenterally-acceptable diluent or solvent or cosolvent or complexing agent or dispersing agent or excipient or combination thereof, for example 1,3-butanediol, polyethylene glycols, polypropylene glycols, ethanol or other alcohols, povidones, various brands of TWEEN surfactant, sodium dodecyl sulfate, sodium deoxycholate, dimethylacetamide, polysorbates, poloxamers, cyclodextrins, lipids, and excipients such as inorganic salts (e.g., sodium chloride), buffering agents (e.g., sodium citrate, sodium phosphate), and sugars (e.g., saccharose and dextrose). Among the acceptable vehicles and solvents that may be employed are water, dextrose solutions, Ringer's solutions and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Depending on the condition being treated, these pharmaceutical compositions may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in the latest edition of “Remington's Pharmaceutical Sciences” (Mack Publishing Co, Easton Pa.). Suitable routes may, for example, include oral or transmucosal administration; as well as parenteral delivery, including intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. For injection, the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. For tissue or cellular administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

The compounds of the present invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.

For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the compounds of the present invention are employed. (For purposes of this application, topical application shall include mouthwashes and gargles).

The pharmaceutical composition and method of the present invention may further comprise other therapeutically active compounds as noted herein which are usually applied in the treatment of the above mentioned pathological conditions. Examples of other therapeutic agents include the following: cyclosporins (e.g., cyclosporin A), CTLA4-Ig, antibodies such as ICAM-3, anti-IL-2 receptor (Anti-Tac), anti-CD45RB, anti-CD2, anti-CD3 (OKT-3), anti-CD4, anti-CD80, anti-CD86, agents blocking the interaction between CD40 and gp39, such as antibodies specific for CD40 and/or gp39 (i.e., CD154), fusion proteins constructed from CD40 and gp39 (CD40Ig and CD8gp39), inhibitors, such as nuclear translocation inhibitors, of NF-kappa B function, such as deoxyspergualin (DSG), cholesterol biosynthesis inhibitors such as HMG CoA reductase inhibitors (lovastatin and simvastatin), non-steroidal antiinflammatory drugs (NSAIDs) such as ibuprofen and cyclooxygenase inhibitors such as rofecoxib, steroids such as prednisone or dexamethasone, gold compounds, antiproliferative agents such as methotrexate, FK506 (tacrolimus, Prograf), mycophenolate mofetil, cytotoxic drugs such as azathioprine and cyclophosphamide, TNF-a inhibitors such as tenidap, anti-TNF antibodies or soluble TNF receptor, and rapamycin (sirolimus or Rapamune) or derivatives thereof.

Other agents that may be administered in combination with invention compounds include protein therapeutic agents such as cytokines, immunomodulatory agents and antibodies. As used herein the term “cytokine” encompasses chemokines, interleukins, lymphokines, monokines, colony stimulating factors, and receptor associated proteins, and functional fragments thereof. As used herein, the term “functional fragment” refers to a polypeptide or peptide which possesses biological function or activity that is identified through a defined functional assay.

The cytokines include endothelial monocyte activating polypeptide II (EMAP-II), granulocyte-macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF), macrophage-CSF (M-CSF), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-12, and IL-13, interferons, and the like and which is associated with a particular biologic, morphologic, or phenotypic alteration in a cell or cell mechanism.

When other therapeutic agents are employed in combination with the compounds of the present invention they may be used for example in amounts as noted in the Physician Desk Reference (PDR) or as otherwise determined by one having ordinary skill in the art.

In the treatment or prevention of conditions which involve compromised vasculostasis an appropriate dosage level can generally be between about 0.01 and about 500 mg per 1 kg of patient body weight per day which can be administered in single or multiple doses. For example, the dosage level can be between about 0.01 and about 250 mg/kg per day; more narrowly, between about 0.5 and about 100 mg/kg per day. A suitable dosage level can be between about 0.01 and about 250 mg/kg per day, between about 0.05 and about 100 mg/kg per day, or between about 0.1 and about 50 mg/kg per day, or about 1.0 mg/kg per day. For example, within this range the dosage can be between about 0.05 and about 0.5 mg/kg per day, or between about 0.5 and about 5 mg/kg per day, or between about 5 and about 50 mg/kg per day. For oral administration, the compositions can be provided in the form of tablets containing between about 1.0 and about 1,000 mg of the active ingredient, for example, about 1.0, about 5.0, about 10.0, about 15.0, about 20.0, about 25.0, about 50.0, about 75.0, about 100.0, about 150.0, about 200.0, about 250.0, about 300.0, about 400.0, about 500.0, about 600.0, about 750.0, about 800.0, about 900.0, and about 1,000.0 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. The compounds can be administered on a regimen of 1 to 4 times per day, such as once or twice per day. There may be a period of no administration followed by another regimen of administration. Preferably, administration of the compound is closely associated with the schedule of IL-2 administration. For example, administration can be prior to, simultaneously with or immediately following IL-2 administration.

It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

Compounds of the present invention can be used, alone or in combination with an effective amount of a therapeutic antibody (or therapeutic fragment thereof), a chemotherapeutic or an immunotoxic agent, for treatment of tumors. While doxorubicin, docetaxel, or taxol are described in the present application as illustrative examples of chemotherapeutic agents, it should be understood that the invention includes combination therapy including a compound of the invention, including but not limited to vasculostatic agents, such as tyrosine, serine or threonine kinase inhibitors, for example, Src-family inhibitors, and any chemotherapeutic agent or therapeutic antibody.

C. EXAMPLES

The following examples are provided to further illustrate the advantages and features of the present invention, but are not intended to limit the scope of the invention.

1. General Methodology

All experiments were performed under anhydrous conditions (i.e. dry solvents) in an atmosphere of argon, except where stated, using oven-dried apparatus and employing standard techniques in handling air-sensitive materials. Aqueous solutions of sodium bicarbonate (NaHCO3) and sodium chloride (brine) were saturated.

Reverse-phase HPLC chromatography was carried out on Gilson 215 liquid handler equipped with Waters SymmetryShield™ RP18 7 μm (40×100 mm) Prep-Pak cartridge. Mobile phase consisted of standard acetonitrile (ACN) and DI Water, each with 0.1% TFA added. Purification was carried out at a flow rate of 40 mL/min, and a gradient such that the peak of interest was eluted between 12-15 min in a 30 min run.

Whereever Suzuki coupling was used, the following general procedure was employed. To a mixture of boronic acid, aryl bromide, and palladium tetrakis(triphenylphosphine)palladium(0) (Pd(Ph3)4) in 4:1 DME/EtOH was added a 2 M aqueous solution of sodium carbonate. The mixture was flushed with argon for 5 min and a condenser was added under argon flow. The reaction was heated to reflux (ca. 100° C.) for 2-18 h. The crude mixture was filtered and the solid filter cake was rinsed thoroughly with MeOH and DCM. The filtrate was concentrated in vacuo and purified by column chromatography.

Whereever Buchwald coupling was used, the following general procedure was employed. A mixture of amine, bromide, Cs2CO3, Xantphos, and Pd2(dba)3 in dioxane was purged with argon for 5 min after which a condenser was added under argon flow. The reaction was heated to reflux (ca 110° C.) for 4-18 h. The crude mixture was filtered and the solid filter cake was rinsed thoroughly with MeOH and DCM. The filtrate was concentrated in vacuo and purified by column chromatography.

Whereever deprotection of aryl methoxy was necessary, the following general procedure was employed. To a solution of the aryl methoxy compound in DCM was added BBr3 (either 1 M in DCM or neat). The reaction was checked for completion and additional BBr3 was added if needed. The reaction was quenched with NaHCO3 and basified to ca. pH 10-11. The biphasic mixture was filtered and the collected solid was rinsed with water. Trace solvents were removed in vacuo.

In general, one of three methods A, B, or C, can be used for synthesizing some of the compounds of the present invention. Those having ordinary skill in the art can determine, depending on variety of factors, including the particular compound that is sought to be made, whether to selected the method A, B or C.

The synthetic method A is shown by the reaction scheme (II). About 1 equivalent of compound 1 was mixed with 2 equivalent of cynamide in a vial. The mixture was heated to about 100° C. until the mixture was completely melted. The mixture was cooled down to room temperature and concentrated HCl was added. The mixture was then again heated at about 100° C. for about 40 minutes and cooled down in ice water. About 14 moles of NaOH were carefully added to the above reaction mixture followed by heating the mixture at about 100° C. for about 2 hours, and by cooling down to room temperature.

Compound 2, shown by the reaction scheme (II), was isolated by filtration and washed several times with water, methanol and diethyl ether. About 1 equivalent of compound 2 was dissolved in N,N-dimethylacetamide, and ethanol solution containing about 4 equivalent of boronic acid and the aqueous solution containing about 0.15 equivalent of potassium carbonate were added. About 0.1 equivalent of triphenylphosphine and about 0.0246 equivalent of tris(dibenzyllideneatone)dipalladium (0) were added to the mixture. The mixture was reflux overnight. The crude product was poured into saturated NaHCO3 solution, and CH2Cl2 was used to extract the product.

Compound 3, shown by the reaction scheme (II), was isolated by removing solvent in the organic phase. Compound 3 was dissolved in N,N-dimethylacetamide in a vial with a septum. Catalytic amount of 10% palladium on carbon was added to the mixture. A balloon filled with hydrogen was placed on the top of the vial. The mixture was stirred at room temperature for about 2 hours. Celite was used to remove the palladium and carbon. Solvent was removed under vacuum and compound 4, shown by the reaction scheme (II), was isolated. About 1 equivalent of compound 4 was dissolved in anhydrous toluene solution containing about 1.2 equivalent of bromide R3Br, about 0.025 equivalent of Pd(dba)3, about 0.07 equivalent of BINAP, and about 0.6 equivalent of KOt-Bu. The mixture was kept at about 100° C. for 24 hours under argon. Compound 5, shown by the reaction scheme (II), was then isolated by high pressure liquid chromatography (HPLC).

The synthetic method B is shown by the reaction scheme (III). Compounds 1, 2, 3, and 4 were consecutively prepared and isolated as described in Method A. About 1 equivalent of compound 4 was dissolved in an aniline followed by adding about 2 equivalents of sulfamic acid. The mixture was heated at about 200° C. overnight. Compound 5 was isolated by HPLC.

The synthetic method C is shown by the reaction scheme (IV). Compounds 1, 2, 3, and 4 were consecutively prepared and isolated as described in Method A. About 1 equivalent of compound 4 was dissolved in substituted phenylamine followed by adding about 2 equivalent of sulfamic acid. The mixture was heated at about 200° C. overnight. Compound 5 was isolated by HPLC and was dissolved in dry CH2Cl2. The mixture was cooled to about −78° C. using a dry ice-acetone bath. About 2 equivalents of BBr3 (1M solution in CH2Cl2) was added dropwise to the mixture at about −78° C. under nitrogen atmosphere. The mixture was then allowed to warm to about 0° C. and stirred overnight at about 0° C., followed by adding saturated aqueous solution of NaHCO3 at about 0° C. and by separating the mixture in a separator funnel. The water layer was extracted twice with CH2Cl2, and the combined organic layer was washed with brine and dried over Na2SO4. Compound 6 was isolated by removing solvent under vacuum. Compound 6 was dissolved in acetone, and about 6 equivalent of K2CO3 and about 2 equivalent of R3Cl were added to the mixture. The mixture was heated to reflux and stirred overnight, cooled and water was added. Compound 7 was isolated by prep HPLC.

Example 1Synthesis of 7-bromo-benzo[1,2,4]triazin-3-ylamine-1-oxide

About 2.48 g (11.4 mmol) of 4-bromo-2-nitro-phenylamine was mixed with about 1.51 g (about 36 mmol) of cynamide in a 20 ml vial. The mixture was heated to about 100° C. till the mixture was totally melted. The mixture was cooled down to room temperature and about 6.5 ml of concentrated HCl was added. The mixture was heated at about 100° C. for about 40 minutes and cooled down in ice water. About 6.5 ml of 14M NaOH was carefully added to the above reaction mixture. The resulted mixture was heated at about 100° C. for about 2 hours, then cooled down to room temperature, and filtrated. After filtration, the precipitate was washed several times with water, methanol and diethylether to remove the starting material. About 0.739 g of the product having formula (V) was obtained. Yield: about 27%. ESI-MS: [M+H]+, 241, 243; 1H NMR (DMSO-d6): δ 7.48 (d, J=9.0 Hz, 1 H), 7.89 (dd, J=9.0 Hz, J2=2.1 Hz, 1 H), 8.26 (d, J=2.1 Hz, 1 H).

About 1 g (4.33 mmol) of 4-bromo-2-methyl-6-nitro-phenylamine was mixed with about 0.5 g (12 mmol) of cynamide and about 5 g pyridine hydrochloride in a 20 ml vial. The mixture was heated to reflux overnight. The mixture was cooled down to room temperature and 10% NaOH was carefully added. The resulted mixture was heated at about 100° C. for about 2 hours, then cooled down to room temperature, and filtrated. After filtration, the precipitate was washed several times with water, acetone and diethyl ether to remove the starting material. About 0.4 g of the product having the formula (VI) was obtained. Yield: about 36%. ESI-MS: [M+H]+, 255, 257; 1H NMR (DMSO-d6): δ 2.45 (s, 3 H), 7.81 (s, 1 H), 8.26 (s, 1 H).

Example 3Synthesis of 7-bromo-5-methyl-benzo[1,2,4]triazin-3-ylamine

About 4.26 g of 7-bromo-5-methyl-benzo[1,2,4]triazin-3-ylamine-1-oxide (VI) prepared as described in Example 2, was dissolved in about 100 ml of acetic acid, and about 1 g of iron powder was added to the solution. The mixture was refluxing for about 10 minutes. The iron was removed by filtration. The solvent in the filtrate was removed under vacuum. Water was added to remove the salt from the product. About 3.9 g of the pure product having formula (VII) was obtained.

Example 4Synthesis of 1-(3-chloropropanyl)pyrrolidine

To the solution of about 186 g (1.18 mol) of 1-bromo-3-chloropropane in about 200 ml of ether was added about 2 equivalent of pyrrolidine at about 0° C. After addition, the mixture was allowed to warm to room temperature and stirred overnight. The resulting white solid residue was removed and to the clear solution was added ice-cold 10% hydrochloric acid. The ether layer was discarded and the acid layer was basified with ice-cold 20% NaOH, extracted with ether and dried over Na2SO4. Ether was removed and the residue was distilled under vacuum (95° C./30 mmHg). About 91 g of product having formula (VIII) was obtained in a form of a pale yellow liquid. The yield was about 53%.

To a solution of about 100 mg (0.42 mmol) of 7-bromo-benzo[1,2,4]triazin-3-ylamine-1-oxide dissolved in about 6 ml of N,N-dimethylacetamide in a 20 ml vial, were added about 240 mg (1.6 mmol) of 2,6-dimethylphenylboronic acid dissolved in about 1 ml of ethanol and about 64 mg (0.6 mmol) of potassium carbonate dissolved in about 1 ml of water. About 9 mg (0.034 mmol) of triphenylphosphine and about 9 mg (9.83 mmol) of tris(dibenzylideneacetone)dipalladium (0) were added to the mixture. The mixture was reflux overnight. The crude product was poured into about 50 ml of saturated aqueous solution of NaHCO3, and CH2Cl2 was used to extract the product. Solvent in the organic phase was removed under vacuum. The residue was dissolved in a mixture of about 2 ml of N,N-dimethylacetamide and about 1 ml of ethyl alcohol in a 20 ml vial with a septum. Catalytic amount of 10% palladium on carbon was added to the mixture. A balloon filled with hydrogen was placed on the top of the vial. The mixture was stirred at room temperature for about 2 hours. Celite was used to remove the palladium and carbon. The crude product and about 200 mg (0.74 mmol) of 1-[2-(4-bromo-phenoxy)-ethyl]-pyrrolidine were dissolved in 10 ml of toluene. About 17 mg (0.018 mmol) of Pd(dba)3, about 34 mg (0.054 mmol) of BINAP, and about 50 mg (0.226 mmol) of KOt-Bu were added to the solution. The mixture was kept at about 100° C. for about 24 hours under argon. The crude product was purified by preparative HPLC. About 2 mg of [7-(2,6-dimethyl-phenyl)-benzo[1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (product having formula (XI)) was isolated. Yield: about 1.6%; ESI-MS: [M+H]+, 440.6; 1H NMR (DMSO-d6): δ 1.90-2.05 (m, 10 H), 3.15 (m, 2 H), 3.59-3.64 (m, 4 H), 4.31 (t, J=5.15 Hz, 2 H), 7.09(d, J=9.06 Hz, 2 H), 7.18 (d, J=7.4 Hz, 2 H), 7.23 (m, 1 H), 7.70 (d, J=8.7 Hz, 1 H), 7.78 (d, J=8.7 Hz, 1 H), 7.90 (d, J=9.06 Hz, 2 H), 8.08 (s, 1 H).

Example 8Synthesis of [7-(2,6-dimethyl-phenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-quinolin-8-yl-amine by Method A

4-(4-bromo-benzoyl)-piperazine-1-carboxylic acid tert-butyl ester (1.5 equiv, 0.49 mmol), 7-(2,6-dichloro-phenyl)-5-methyl-benzo[1,2,4]triazin-3-ylamine (1.0 equiv, 0.33 mmol), Xantphos (0.1 equiv, 0.123 mmol), palladium acetate (0.05 equiv, 0.0165 mmol), and Potassium-t-butoxide (2.0 equiv, 0.66 mmol) were dissolved in 10 mL of dioxane, purged of air and placed under an argon blanket before refluxing at 100° C. for 18 h. The reaction was cooled to room temperature and filtered before extracting with EtOAc and washing with saturated bicarbonate and brine, followed by drying over Na2SO4. The organics were condensed under reduced pressure to give brown oil that was precipitated out using EtOAc/hexanes (1:5 v/v) to afford brown/tan precipitate. BOC protecting group was removed with 30% TFA/DCM (v/v) stirring at room temperature for 1 h.

1-(2-chloroethyl)pyrrolidine hydrochloride (1.0 equiv., 8.82 mmol) and 3-bromothiophenol (1.5 equiv., 13.23 mmol) were dissolved in acetonitrile (100 mL). Potassium carbonate (10.0 equiv., 88.2 mmol) was added to the reaction while stirring. The reaction was kept in oil bath at 80° C. to reflux for 18 h. The reaction was then cooled to room temperature and filtered to remove excess inorganics, and the organics were extracted with saturated NaHCO3 and brine. The organics were dried over Na2SO4 then concentrated under reduced pressure. The resulting crude oil was purified by flash chromatography using 4:6 EtOAc/hexanes. The fractions were concentrated to afford product as a yellow oil (792.3 mg, 32% yield). Rf=0.75 MS (ES+): m/z=287 LC retention time: 1.81 min.

7-(2-chloro-5-methoxy-phenyl)-6-methyl-benzo[1,2,4]triazin-3-ylamine (1.0 equiv., 0.28 mmol), 4-bromo-N-methyl-N-(2-pyrrolidin-1-yl-ethyl)-benzenesulfonamide (1.5 equiv., 0.42 mmol), Cs2CO3 (3.0 equiv., 0.84 mmol), Pd2(dba)3 (0.1 equiv., 0.028 mmol), and Xantphos (0.2 equiv., 0.056 mmol) were dissolved in 20 mL of dioxane and argon was bubbled through the reaction mixture. The reaction was kept in oil bath to reflux at 100° C. for 18 h. The reaction was then cooled to room temperature, filtered to remove inorganics, extracted with EtOAc, saturated NaHCO3 and brine, and the organics were dried over Na2SO4, then concentrated under reduced pressure to give an orange solid (147.2 mg, 93% yield). The resulting crude material was used as is to prepare 4-[7-(2-chloro-5-hydroxy-phenyl)-6-methyl-benzo[1,2,4]triazin-3-ylamino]-N-methyl-N-(2-pyrrolidin-1-yl-ethyl)-benzenesulfonamide as described in the next example.

4-[7-(2,6-dimethyl-phenyl)-5-nitro-1-oxy-benzo[1,2,4]triazin-3-ylamino]-N-(2-pyrrolidin-1-yl-ethyl)-benzenesulfonamide (0.079 g, 0.14 mmol, 1 equiv) was combined with 10% palladium on carbon (0.029 g) and flushed with argon. The reactants were then diluted with methanol (5 mL) and the reaction atmosphere was evacuated and replaced with hydrogen. A hydrogen balloon was affixed and the reaction was allowed to stir for 3 hours. Argon was then bubbled through the reaction mixture and contents were filtered though a pad of Celite. Solvents were evaporated to provide a green solid product (0.045 g, 62% yield). MS (ESI+): 518.4 (M+H), r.t.=2.56 min.

7-(2-chloro-5-methoxy-phenyl)-5-methyl-benzo[1,2,4]triazin-3-ylamine (0.158 g, 0.527 mmol, 1 equiv) was diluted with 10 mL DCM and chilled to 0° C. using an ice bath. A 1.0 M solution of BBr3 in DCM (3.16 mL, 3.16 mmol, 6.0 equiv) was then added in one portion resulting in a dark reaction mixture. The reaction was allowed to come to ambient temperature and stirred for 3.5 hours, then quenched by carefully pouring onto a saturated solution of sodium bicarbonate followed by sonication for 3-5 minutes. Additional DCM was added and this mixture was washed with sodium bicarbonate followed by brine. The organic phase was separated, dried over sodium sulfate (Na2SO4), filtered and evaporated to dryness. Solids were then diluted with minimum amount of ethyl acetate and precipitated out with hexanes. Product was filtered off and dried to yield a yellow solid (0.110 g, 73% yield). MS (ESI+): m/z 287.1, LC retention time: 2.46 min.

The title compound was synthesized as shown by the reaction scheme (CXXVI).

To a solution of the 7-bromo-5-methylbenzo[1,2,4]triazin-3-yl-amine-1-oxide 1 shown on scheme (CCXXVI) (100 mg, 0.39 mmol) in N,N-dimethylacetamide (6 mL) was added a solution of 4-methoxy-2,6-dimethylphenylboronic acid 2 (210 mg, 1.17 mmol) in EtOH (1 mL), a solution of K2CO3 (90 mg, 0.65 mmol) in H2O (1 mL), tris(dibenzylidenacetone)dipalladium [0] (Pd2 (dba)3, 9 mg, 0.01 mmol), and PPh3 (9 mg, 0.034 mmol). The suspension was heated under reflux for 18 h. The cold reaction mixture was poured into saturated NaHCO3 and CH2Cl2 was used to extract the product. The crude product was purified by chromatography (SiO2/Hexane:EtOAc=1:1) to afford 3 as yellow solid. The 3 was reduced to 4 by hydrogenation, as shown by scheme (CCXXVI). To a solution of 4 in CH2Cl2 was added 1.0 M BBr3 in CH2Cl2 (1 mL, 1 mmol). The mixture was stirred overnight at room temperature. The saturated NaHCO3 was added and the organic layer was separated. The aqueous layer was extracted with CH2Cl2 (3×10 mL). The combined organic solution was dried (MgSO4) to afford 5.

The title compound was synthesized as shown by the reaction scheme (CXXXVII).

To a solution of 11 shown by scheme (CXXXVII) (646 mg, 2.44 mmol) in dry DMF (20 mL) was added ethyl 2-bromothiazole-4-carboxylate 18 (750 mg, 3.17 mmol), rac-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP, 304 mg, 0.48 mmol), Pd2 (dba)3 (223 mg, 0.24 mmol), and Cs2CO3 (1.6 g, 4.88 mmol). The mixture was heated under reflux for 20 h. The solid was filtered off and washed with DMF. The filtrate was concentrated. The residue was dissolved in CH2Cl2 and washed with brine (3×100 mL). The crude product ethyl 2-(5-methyl-7-(2,6-dimethylphenyl)benzo[e][1,2,4]triazin-3-ylamino)thiazole-4-carboxylate 19 was dissolved in MeOH (10 mL) and THF (10 mL), followed by adding solution of LiOH (586 mg, 24.4 mmol) in H2O (10 mL). The mixture was heated at 60° C. for 2 h. The solvent was removed in vacuo. The product was purified using chromatograpy (SiO2/CH2Cl2:MeOH:NH3.H2O=100:10:2.5 and then 100:100:50). 2-(5-methyl-7-(2,6-dimethylphenyl)benzo[e][1,2,4]triazin-3-ylamino)thiazole-4-carboxylic acid (750 mg, 78%) (compound 20 shown by scheme (CXXXVII)) was obtained as an orange solid.

The synthesis was continued by dissolving compound 20 (400 mg, 1.02 mmol) in anhydrous CH2Cl2 (20 mL), followed by adding 2-chloro-4,6-dimethoxy-1,3,5-trizine (CDMT, 198 mg, 1.12 mmol) and 4-methylmophline (NMM, 0.23 mL, 2.04 mmol). The mixture was stirred at room temperature for 0.5 h, followed by adding 2-(pyrrolidin-1-yl)ethanamine 21 (198 mg, 1.12 mmol). The reaction was stirred at room temperature overnight. The solid was filtered off and washed with CH2Cl2. The filtrate was washed with saturated NaHCO3 (1×50 mL) and then brine (2×50 mL). The organic solution was dried (Na2SO4).

The title compound was synthesized as shown by the reaction scheme (CXXXIX).

To a solution of ethyl formate (10 mL) in anhydrous Et2O was added compound 21 as shown on scheme (CXXXIX) (2 mL). The solution was stirred at room temperature for 2 h. The solvent was removed in vacuo, followed by adding 1.0 M LiAlH4 in THF (5 mL). The mixture was heated under reflux overnight and cooled. Water was then added slowly. The solid was filtered off and washed with THF. The filtrate was concentrated and remained aqueous, and was extracted with Et2O (3×20 mL) and dried (Na2SO4). The solvent was removed and the residue was dissolved in CH2Cl2 (10 mL), followed by adding 4-bromobenzenesulfonyl chloride 24 (1.2 g, 4.72 mmol) and Et3N (3 mL). The mixture was stirred at room temperature for 3 hours, then saturated NaHCO3 (100 mL) was added. The organic layer was separated and the aqueous was extracted with CH2Cl2 (2×10 mL). Combined organic solution was dried (Na2SO4). The solvent was removed in vacuo and afforded compound 25 shown on scheme (CXXXIX) as a yellow oil.

The title compound was synthesized as shown by the reaction scheme (CXLIX).

Compound 33 shown by scheme (CXLIX) was prepared by a method that was analogous to that used to make compound 30 as shown on scheme (CXLI), except 2-chloro-5-methoxyphenylboronic acid (compound 31) (510 mg, 2.73 mmol) and 7-bromo-5-methylbenzo[e][1,2,4]triazin-3-amine (compound 32) (503 mg, 2.10 mmol) were used to produce compound 33 (500 mg, 80%) as a yellow solid. The title compound (CXLVIII) was then prepared by a method that was analogous to that used to make compound (CXL) as described in Example 128, except compounds 33 (500 mg, 1.66 mmol) and 6 (674 mg, 2.49 mmol) were used as shown by scheme (CXLIX) to afford the title compound (CXLVIII) (440 mg, 54%) as a yellow solid.

To a solution of compound 48 (47 mg, 0.15) in Me2CO3 (20 mL) were then added Cs2CO3 (202 mg, 0.62 mmol), 1-(2-chloroethyl)pyrrolidine 43 (26.4 mg, 0.15 mmol), and NaI (15 mg, 0.1 mmol). The mixture was heated under reflux overnight. The solid was filtered off and washed with Me2CO. The filtrate was concentrated in vacuo. The residue was dissolved in CH2Cl2 and washed with brine (2×50 mL). The organic layer was separated and dried (Na2SO4). The solvent was removed in vacuo and compound 49 was obtained as a yellow solid. Compound 51 was then prepared by using a method that was analogous to that used to synthesize compound 39 (scheme (CLIV), Example 136), except compound s 49 (62 mg, 0.15 mmol) and 50 (3-bromopyridine) (36.7 mg, 0.23 mmol) were used to afford compound 51 as a yellow solid.

The title compound was synthesized as shown by the reaction scheme (CLXII).

As shown by scheme (CLXII), the solution of 4-bromo-1H-indole 52 (1.56 g, 7.96 mmol) in anhydrous Et2O (20 mL) was cooled in ice-H2O, followed by adding NaH (0.22 g, 9.6 mmol). The suspension was stirred at 0° C. for 30 min. The flask was further cooled in dry ice-acetone and 2.5 M BuLi in hexane (8 mL, 20 mmol) was dropped. After stirring for 30 min, the neat B(OMe)3 was dropped and the mixture was stirred at room temperature overnight. The mixture was put into 1 M H3PO4 in portions and stirred for 30 min. The organic layer was separated and the aqueous layer was extracted with Et2O (2×20 mL). The combined organic layer was washed with 1 M NaOH (3×50 mL). The combined aqueous was actified with 1 M H3PO4 (pH<2) and extracted with Et2O (3×20 mL). The combined organic solution was dried (Na2SO4). The solvent was removed and compound 53 (1.16 g, 91%) was obtained, as demonstrated by scheme (CLXII), as a wax solid.

The title compound was synthesized as shown by the reaction scheme (CLXV).

The intermediate compounds 57 and 58 shown on scheme (CLXV) were prepared as follows. Compound 57 was prepared by using a method that was analogous to that used to synthesize compound 30 shown on scheme (CXLI) and described in Example 128, except 2-bromo-5-methoxyphenylboronic acid (compound 56) (233 mg, 1.1 mmol) and 7-bromo-5-methylbenzo[e][1,2,4]triazin-3-amine (compound 32) (240 mg, 1.0 mmol) were used to afford compound 57 (300 mg, 87%) as a yellow solid. Compound 58 was prepared by using a method that was analogous to that used to synthesize compound (CXL), as described in Example 128, except compounds 57 (150 mg, 0.43 mmol) and 54 (174 mg, 0.52 mmol) were used, as shown on scheme (CLXV) to afford compound 58 as a yellow solid.

The title compound was synthesized as shown by the reaction scheme (CLXVII).

As shown by scheme (CLXVII), compound 60 was prepared by using a method that was analogous to that used to synthesize compound 30 shown on scheme (CXLI) and described in Example 128, except 2-chloro-6-methoxyphenylboronic acid 59 (1 g, 4.18 mmol) and 7-bromo-5-methylbenzo[e][1,2,4]triazin-3-amine 32 (1.2 g, 6.28 mmol) were used to give compound 60 (7-(2-chloro-6-methoxyphenyl)-5-methylbenzo[e][1,2,4]triazin-3-amine) (1.12 g, 90%) as a yellow solid. Compound 61 was prepared was prepared by using a method that was analogous to that used to synthesize compound (CXL), as described in Example 128, except compounds 60 (390 mg, 1.30 mmol) and 54 (518 mg, 1.56 mmol) were used to afford compound 61 (4-(7-(2-chloro-6-methoxyphenyl)-5-methylbenzo[e][1,2,4]triazin-3-ylamino)-N-(2-(pyrrolidin-1-yl)ethyl)benzensulfonamide) (680 mg, 95%) as a yellow solid.

To synthesize the title compound (CLXIX), two intermediate compounds 62 (2-[4-(6-chloro-2-methyl-pyrimidin-4-yl)-piperazin-1-yl]-ethanol) and 63 (7-(2,6-dichloro-phenyl)-5-methyl-benzo[1,2,4]triazin-3-ylamine) shown below were used.

To synthesize compound 62, to a solution of 4,6-dichloro-2-methyl-pyrimidine (5.0 g, 31 mmol) and 2-piperazin-1-yl-ethanol (2.7 g, 21 mmol) in dioxane (25 mL) was added DIPEA (3.0 mL, 17 mmol). The mixture was heated at reflux for 16 h. The mixture was allowed to cool to room temperature and poured into water. The reaulting aqueous layer was extracted with EtOAc and the combined organic layers washed with brine, dried over Na2SO4 and filtered. The filtrate was concentrated and the residue purified by flash chromatography on silica gel (5-10% MeOH/DCM) to afford compound 62 as a brown liquid (2.1 g, 39%). MS (ESI+): m/z 257.

To synthesize the title compound (CLXX), an intermediate compound 64 (2-(4-{6-[7-(2-chloro-5-methoxy-phenyl)-5-methyl-benzo[1,2,4]triazin-3-ylamino]-2-methyl-pyrimidin-4-yl}-piperazin-1-yl)-ethanol) shown below was used.

To synthesize compound 70, a solution of N,N-diethyl-N′-methyl-ethane-1,2-diamine (3.4 g, 26 mmol), 4-bromobenzyl bromide (5.0 g, 20 mmol) and cesium carbonate (13 g, 40 mmol) in acetonitrile (60 mL) was heated at reflux for 18 h. The mixture was cooled and poured into water. The aqueous layer was extracted with EtOAc and the combined organic layers washed with brine, dried over Na2SO4, and filtered. The filtrate was concentrated and the crude compound 70 was used in the next step without purification.

The synthesis of the title compound (CXCVIII) can be generally described by the sequence of reaction schemes (CXCIX), (CC), (CCI), (CCII), (CCIII), and (CCIV).

To freshly prepared sodium methoxide using sodium (900 mg, 39 mmol) and anhydrous methanol (20 mL) was added 3-chloro-6-nitroaniline (2.55 g, 15 mmol). The reaction mixture was heated in a sealed tube at 120° C. for 4 hours. The solvent was removed under reduced pressure and the crude solid was dissolved in water (50 mL) and extracted with ethyl acetate (3×50 mL). The combined ethyl acetate layer was dried over sodium sulfate. The sodium sulfate was removed by filtration and the solvent was removed. The crude was dried to yield a yellow solid (1.75 g, 69%).

The product of reaction shown by scheme (CXCIX) was further reacted as shown by scheme (CC).

A mixture of 3-methoxy-6-nitroaniline (940 mg, 5.6 mmol) and NBS (1.1 g, 6.2 mmol) in glacial acetic acid (25 mL) was heated to reflux under argon for 3 h. The reaction mixture was brought to room temperature and the mixture was diluted with water (100 mL). The yellow precipitate was collected by filtration, washed with water to yield a yellow solid (1 g, 72%).

The product of reaction shown by scheme (CC) was further reacted as shown by scheme (CCI).

4-bromo-3-methoxy-6-nitroaniline (1 g, 4.05 mmol) was mixed with cyanamide (1.7 g, 40 mmol) and heated to melt at 90° C. Concentrated HCl (10 mL) was cautiously added dropwise within 20 minutes. The reaction mixture was heated to reflux until all starting material reacted (about 2 h). Another batch of cyanamide (1.7 g, 40 mmol) and hydrochloric acid (10 mL) was added and the reaction continued for another hour. The ice cooled reaction mixture was brought to pH 13 with 30% sodium hydroxide. The mixture was heated to reflux for 3 h. The precipitate was collected by filtration and washed with water to give a yellow solid (900 mg, 82%).

The product of reaction shown by scheme (CCI) was further reacted as shown by scheme (CCII).

The product of reaction shown by scheme (CCVIII) was further reacted as shown by scheme (CCIX).

3-amino-7-o-tolylbenzo[e][1,2,4]triazin-6-ol-1-N-oxide (60 mg, 0.2 mmol) was reduced in methanol (10 mL) and ethyl acetate (10 mL) with catalytic amount of Raney Ni and hydrogen at room temperature for 4 hours. The catalyst was removed by filtration and the solvent was removed. The solid was washed with acetone to give a yellow solid (50 mg, 89%).

The product of reaction shown by scheme (CCIX) was further reacted as shown by scheme (CCX).

The product of reaction shown by scheme (CCXIV) was further reacted as shown by scheme (CCXV).

4-[7-(3-benzyloxy-phenyl)-6-methoxy-1-oxy-benzo[1,2,4]triazin-3-ylamino]-N-(2-pyrrolidin-1-yl-ethyl)-benzenesulfonamide (0.1 g, 0.16 mmol) was dissolved in methanol (10 mL) and ethyl acetate (10 mL) with catalytic amount of Raney Ni and atmosphere hydrogen at room temperature for 4 hours. The catalyst was removed by filtration. The solvent was removed by reduced pressure. The solid was used for the next reaction without further purification.

The product of reaction shown by scheme (CCXV) was further reacted as shown by scheme (CCXVI).

The synthesis of the title compound (CCXXVI) can be generally described by the sequence of reaction schemes (CCXXVII), (CCXXVIII), (CCXXIX), and (CCXXX).

2.5M nBuLi (14.9 mL, 37.4 mmol) was added dropwise over 5 minutes into a mixture of 4-chloro-2-fluoro-1-methoxybenzene (5 g, 31.1 mmol) in anhydrous THF (30 mL) at −78° C. After the mixture was stirred at −78° C. for 20 minutes, anhydrous trimethyl borate (4.9 g, 46.6 mmol) was added into the solution at −78° C. The reaction mixture was brought to room temperature for over period of 2 h. The reaction was quenched by 2N HCl (1 mL). THF was removed by vacuum. The crude product was diluted with 2N HCl (100 mL). The acidic solution was extracted with ethyl acetate (2×50 mL). The combined ethyl acetate fraction was dried over sodium sulfate. The sodium sulfate was removed by filtration and the solvent was removed by vacuum. The oil was titrated with hexanes/chloroform (1:1) to yield a solid. The precipitate was collected by filtration and washed with hexanes to yield a white solid (2.5 g, 39%). 1H NMR DMSO-d6: δ 3.81 (s, 3H), 7.12 (m, 2H), 8.66 (s, 2H).

The product of reaction shown by scheme (CCXXVII) was further reacted as shown by scheme (CCXXVIII).

The synthesis of the title compound (CCXXXI) can be generally described by the sequence of reaction schemes (CCXXXII), (CCXXXIII), (CCXXXIV), (CCXXXV), and (CCXXXVI).

A solution of 5-methoxy-2-methylbenzenamine (1.3 g, 9.48 mmol) in 48% HBr (13 mL) and EtOH (10 mL) was cooled to 0° C. and NaNO2 (0.78 g, 11.30 mmol) in water (5 mL) was added dropwise over 15 minutes while keeping the temperature of the reaction between 0-5° C. After the clear pale brown solution was kept at 0° C. with stirring for 1 h, this solution was transferred into the boiling solution of CuBr (6.8 g, 47.4 mmol) in 48% HBr (25 mL). The solution was refluxed for overnight. The reaction mixture was cooled to room temperature and the reaction was diluted with water (50 mL). The acidic solution was extracted with ethyl acetate (2×50 mL). The combined ethyl acetate was washed by saturated NaCl (1×50 mL) and was dried over sodium sulfate. The sodium sulfate was removed by filtration and the solvent was removed by vacuum. The crude product was purified by silica gel column (3.5×16 cm) chromatography with hexanes as an eluent to yield colorless oil (750 mg, 39%).

The product of reaction shown by scheme (CCXXXII) was further reacted as shown by scheme (CCXXXIII).

2.5M nBuLi (1.8 mL, 4.5 mmol) was added dropwise over 5 minutes into a mixture of 2-bromo-4-methoxy-1-methylbenzene (750 mg, 3.73 mmol) in anhydrous THF (30 mL) at −78° C. After the mixture was stirred at −78° C. for 20 minutes, anhydrous trimethyl borate (0.62 g, 6 mmol) was added into the solution at −78° C. The reaction mixture was brought to room temperature for over period of 2 h. The reaction was quenched by 2N HCl (1 mL). The THF was removed by vacuum. The crude product was diluted with 2N HCl (100 mL). The acidic solution was extracted with ethyl acetate (2×50 mL). The combined ethyl acetate fraction was dried over sodium sulfate. The sodium sulfate was removed by filtration and the solvent was removed by vacuum to yield a pale yellow solid (0.43 g, 69%).

The product of reaction shown by scheme (CCXXXIII) was further reacted as shown by scheme (CCXXXIV).

To a mixture of 3-methylthiophen-2-yl-2-boronic acid (252 mg, 2 mmol), 3-amino-7-bromo-6-methyl benzo-1,2,3-triazine (239 mg, 1 mmol) and Pd(Ph3)4) (58 mg, 0.05 mmol) in 4:1 DME/EtOH was added a 2M aqueous solution of sodium carbonate (4 mL). The mixture was flushed with argon for 5 min and was heated under reflux (ca. 100° C.) for 16 h. The volatiles were evaporated and the residue was triturated with chloroform-water (1:1, 200 mL). The chloroform layer was separated and filtered through a small silica plug. The silica plug was washed with 200 mL of 10% methanol in chloroform. On evaporation the crude product was obtained as a brown solid (255 mg).

To a mixture of 4-methylpyridin-3-yl-3-boronic acid (272 mg, 2 mmol), 3-amino-7-bromo-6-methyl benzo-1,2,3-triazine (239 mg, 1 mmol) and Pd(Ph3)4) (58 mg, 0.05 mmol) in 4:1 DME/EtOH was added a 2M aqueous solution of sodium carbonate (4 mL). The mixture was flushed with argon for 5 min and was heated under reflux (ca. 100° C.) for 16 h. The volatiles were evaporated and the residue was triturated with chloroform-water (1:1, 200 mL). The chloroform layer was separated and filtered through a small silica plug. The silica plug was washed with 200 mL of 10% methanol in chloroform. On evaporation the crude product was obtained as a brown solid (260 mg).

To a mixture of 4-chloropyridin-3-yl-3-boronic acid (314 mg, 2 mmol), 3-amino-7-bromo-6-methyl benzo-1,2,3-triazine (239 mg, 1 mmol) and Pd(Ph3)4) (58 mg, 0.05 mmol) in 4:1 DME/EtOH was added a 2M aqueous solution of sodium carbonate (4 mL). The mixture was flushed with argon for 5 min and was heated under reflux (ca. 100° C.) for 16 h. The volatiles were evaporated and the residue was triturated with chloroform-water (1:1, 200 mL). The chloroform layer was separated and filtered through a small silica plug. The silica plug was washed with 200 mL of 10% methanol in chloroform. On evaporation the crude product was obtained as a brown solid (260 mg).

The following Examples 188-192 describe the synthesis of some of the intermediate compounds 100 (7-bromo-5-methyl-benzo[1,2,4]triazin-3-ylamine), 101 (7-bromo-6-methyl-benzo[1,2,4]triazin-3-ylamine), 102 ((4-bromo-phenyl)-(4-methyl-piperazin-1-yl)-methanone), 103 (4-bromo-N-(2-pyrrolidin-1-yl-ethyl)-benzamide), 104 (4-bromo-N-(2-dimethylamino-ethyl)-benzamide), 105 (3-bromo-N-(2-pyrrolidin-1-yl-ethyl)-benzamide), 106 (3R-(3-bromo-benzoylamino)-pyrrolidine-1-carboxylic acid tert-butyl ester), 107 (1-[3-(3-bromo-benzenesulfonyl)-propyl]-pyrrolidine), and 108 (7-(2,6-dimethyl-phenyl)-5-methyl-benzo[1,2,4]triazin-3-ylamine) that were used to synthesize the title compounds of Examples 188-190, 193-205, 208, 209, and 213 that follow.

To a solution of 4-bromo-benzoic acid (5.02 g, 25 mmol) in 200 mL acetonitrile were added N1,N1-dimethylethylenediamine (2.8 mL, 25 mmol) and EDC (4.9 g, 25.5 mmol). The mixture was stirred at room temperature for 2 h. The white precipitate was removed by filtration and the solvent was evaporated. The residue was dissolved in 150 mL CH2Cl2, and was washed successively with water (150 mL) and aqueous saturated NaHCO3 (150 mL). The organic phase was dried (MgSO4) and the solvent was evaporated. The crude product was purified by flash column chromatography Rf=0.2, (CH2Cl2/MeOH, 90:10) to afford the title intermediate compound 104 (3.68 g, 54%). MS (ESI+) m/z=271/273.

The title compound was prepared according to the procedure described for the intermediate compound 104 (Example 189). The crude product was used without further purification (3.7 g, 50%). MS (ESI+) m/z=297/299.

To a solution of 3-bromothiophenol (4.0 g, 21.2 mmol) in 50 mL methanol was added NaOMe (2.28 g, 42 mmol). The mixture was stirred at room temperature for 1 h and was added dropwise to 22 mL of 1,3-dibromopropane (42.5 g, 210 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 h and quenched with water (50 mL). The crude product was extracted with CH2Cl2 (100 mL) and the combined organic phase was dried (MgSO4). The volatiles were removed under reduced pressure. The crude product in 150 mL CH2Cl2 was treated with 3-chloroperoxybenzoic acid (4.9 g, 20 mmol) at 0° C. for 1 h. Another batch of mCPBA (4.9 g, 20 mmol) was added and the stirring was continued for another 30 min at 0° C. before the mixture was allowed to warm to room temperature. The reaction mixture was diluted with CH2Cl2 and EtOAc (20 mL each) and washed twice with saturated aqueous NaHCO3 solution. The organic phase was dried (MgSO4) and the product was purified by silica gel column chromatography (Rf=0.53, EtOAc/hexanes 50:50) to give 1-bromo-3-(3-bromo-propane-1-sulfonyl)benzene as a colorless solid (5.47 g, 76%).

A mixture of 1-bromo-3-(3-bromo-propane-1-sulfonyl)benzene (5.47 g, 16.4 mmol Cs2CO3 (10.7 g, 32.8 mmol) and pyrrolidine (2.71 mL, 32.8 mmol)) in 100 mL anhydrous 1.4-dioxane was stirred at room temperature for 16 h. The reaction mixture was quenched with saturated aqueous sodium bicarbonate (100 mL) and was extracted with CH2Cl2 (140 mL). The combined organic phase was dried (MgSO4), and the solvent was removed. The product was dried in vacuo to afford the title intermediate compound 107, as a brown oil (4.95 g, 91%).

As a preliminary step, the synthesis of the title product included a process shown by the reaction scheme (CCLXIX).

The reaction scheme (CCLXIX) illustrates a synthetic process rthat included dropwise adding of 6.4 g bromine (40 mmol) to a solution of 4-nitrobenzene-1,3-diamine (5.56 g, 36.3 mmol), in 50 mL 1,4-dioxane, followed by stirring the solution was room temperature for 16 h. The yellow precipitate was isolated by filtration, washed with 1,4-dioxane (30 mL) and dried in vacuo to give 4-bromo-6-nitro-benzene-1,3-diamine dihydrobromide salt designated on the reaction scheme (CCLXIX) as compound 13 (8 g, 95%).

A mixture of compound 13 (6.0 g, 15.3 mmol) and cyanamide (3.9 g, 92 mmol) was heated to a melt at 90° C. To the melt was cautiously added concentrated HCl (45 mL) and the reaction mixture was stirred at 95° C. for 2 h. It was cooled to room temperature and the reaction mixture was brought to pH 12 with 30% sodium hydroxide (about 65 mL). The mixture was heated to 90° C. for 2 h, cooled to room temperature and poured into water (1 L). The yellow precipitate was isolated by filtration, washed with water (100 mL) and acetone (20 mL) and was dried to give 7-bromo-1-oxy-benzo[1,2,4]triazine-3,6-diamine designated on the reaction scheme (CCLXIX) as compound 14 (1.3 g, 33%). To a suspension of the crude product in 140 mL MeOH were added Raney Ni and the mixture was hydrogenated for 2 h. Solids were removed by filtration through a short silica gel column. The solvent was evaporated to give 7-bromo-benzo[1,2,4]triazine-3,6-diamine designated on the reaction scheme (CCLXIX) as compound 15 (1.0 g, 82%). To a solution of compound 15 (1.0 g, 4 mmol) in 60 mL DMA were added 2-methylphenylboronic acid (884 mg, 6.5 mmol) in 16 mL ethanol, K2CO3 (332 mg, 2.4 mmol) in 6 mL water, PPh3 (262 mg, 1 mmol) and Pd2(dba)3 (184 mg, 0.2 mmol). The reaction mixture was stirred for 4 h at 100° C. under argon, cooled to room temperature and poured into aqueous saturated NaHCO3 solution (100 mL). The product was extracted with CH2Cl2 (150 mL) and purified by reverse phase preparative HPLC. The product was washed with saturated aqueous NaHCO3 solution and extracted with EtOAc (100 mL). The combined organic phases were dried (Na2SO4) and the solvent removed. The crude product was re-crystallized from MeOH to give 7-o-tolyl-benzo[1,2,4]triazine-3,6-diamine designated on the reaction scheme (CCLXIX) as compound 16 (133.7 mg, 13%).

As a preliminary step, the synthesis of the title product included a process shown by the reaction scheme (CCLXXVI).

To a melt of 5-chloro-2-nitroaniline (5 g, 29 mmol) and cyanamide (9.8 g, 231 mmol) at 100° C. was cautiously added concentrated HCl (50 mL) and the mixture was stirred at 100° C. for 2.5 h. The reaction mixture was cooled to room temperature and the pH was adjusted to pH 11 using 30% aqueous NaOH. The reaction mixture was stirred at 100° C. for 2 h, cooled to room temperature and poured into water (200 mL). The yellow precipitate was isolated by filtration, washed with CH2Cl2 (20 mL) and dried to give 6-chloro-1-oxy-benzo[1,2,4]triazin-3-ylamine, designated on the reaction scheme (CCLXXVI) as compound 17 (3.42 g, 60%). To a solution of compound 17 (983 mg, 5 mmol) in 30 mL glacial acetic acid was added NBS (1.78 g, 10 mmol) and the mixture was heated to reflux for 4 h. It was cooled to room temperature and poured into water (700 mL). The yellow precipitate was isolated by filtration and was hydrogenated in MeOH (100 mL) for 2 h using Raney Ni (1.0 g). Solids were removed by filtration and the solvent was evaporated. The crude product was purified by silica gel column chromatography using EtOAc as the mobile phase to give 7-bromo-6-chloro-benzo[1,2,4]triazin-3-ylamine, designated on the reaction scheme (CCLXXVI) as compound 18 (95.6 mg, 7.4%).

The ability of compounds of the present invention to inhibit the activity of three groups of kinases was tested. Kinases tested included the src family (primarily src and yes), some angiogenic growth factor receptors (Fgfr1, Vegfr, and Pdgfrβ) and the ephrin, EphB4. All kinase reactions were conducted in 96-well plates with a final reaction volume of 50 ul.

Pdgfrβ (0.16 ug/well, Panvera/Invitrogen) 500 nM ATP and the PTK2 peptide (700 uM) were combined with compound and reaction buffer as noted above for src. The reaction was incubated for 60 minutes at 37 C, and the residual ATP concentration was determined using the luciferase-based technique also noted above.

FGFR1 and VEGFR2 kinase assays were similarly performed. FGFR1 (76 ng/well, Panvera/Invitrogen) was combined with 12.5 mg/ml poly(glu4tyr) (Sigma) and 2.5 uM ATP. VEGFR2 (14.1 U/well, Cell Signaling/ProQinase) was used with 0.3 mg/ml poly(glu4tyr) and 1.5 uM ATP. Both were incubated for 60 minutes at 37 C, and the residual ATP was measured via luminescence, per the procedure described above.

EphB4

EphB4 kinase activity was similarly measured, using the luciferase-based technique described above. 28.9 mU/well EphB4 (Upstate) was reacted with 1 mg/ml poly(glu4tyr), 6 uM ATP and test reagents. The reaction was incubated for 60 minutes at 37 C and the residual ATP concentration was measured.

The test results for inhibition of Src kinase are presented in Table 1, and the test results for inhibition of some other kinases (i.e., Yes, EphB4, and Pdgfrβ) are presented in Table 2. The abbreviation “IC50” means that a particular compound of the invention, when present at the specified concentration, inhibited the kinase by 50%.

TABLE 1

Tests Results of Inhibition of Src Kinase by Some Compounds of the Invention